PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is the second most common leukemia in adults and typically has a dismal prognosis and high mortality, which is exemplified by a 28% five-year overall survival rate. Venetoclax, a selective inhibitor of the anti-apoptotic protein BCL-2, has received FDA approval for the treatment of AML. Despite promising early responses of AML patients to venetoclax, drug resistance ensues after prolonged treatment and highlights the urgency for a deep understanding of the underlying mechanisms. Recently, I discovered that mitochondria in AML cells undergo a morphological change upon venetoclax resistance. Using a genome-wide CRISPRi screen in human AML, I identified genes involved in mitochondrial structure as synthetic lethal vulnerabilities for venetoclax in AML. Mitochondria of venetoclax-resistant AML cells actively modify their architecture and function to prevent apoptosis. Supporting this, OPA1, the master regulator of mitochondrial cristae structure, and CLPB, a mitochondrial chaperonin, were strikingly upregulated in venetoclax-resistant AML cells relative to the sensitive cells. CLPB directly interacts with OPA1 to maintain the physiological mitochondrial morphology. Promisingly, genetic CLPB or OPA1 ablation enhances venetoclax-induced apoptosis of AML cells, by promoting cristae remodeling and mitochondrial stress. This proposal aims to leverage these observations by 1) delineating the mechanistic details by which mitochondrial dynamics and homeostasis lead to acquisition of drug resistance in AML, using super-resolution microscopy, electron tomography, and biochemical techniques, and 2) assessing the therapeutic potential of perturbing mitochondrial structure to augment venetoclax action in preclinical AML mouse models. This research stands to have significant clinical impact, because it can serve as a basis for developing new combinational targeted therapies for patients with AML. In addition, this proposal outlines my career development plan for obtaining the requisite training to transition into a successful independent investigator. This includes 1) guidance from my esteemed mentor Dr. Iannis Aifantis, expert in blood malignancies and mouse models; 2) scientific training by an expert advisory panel, consisting of Drs. Raoul Tibes, Hans-Willem Snoeck, Kivanc Birsoy and Evripidis Gavathiotis, all in top institutes of NYC; 3) hands-on training using state-of-art equipment, including super-resolution microscopy with Dr. Eli Rothenberg; 4) collaboration with experts in microscopy and bioinformatics; and 5) career development courses sponsored by NYU. The laboratory of Dr. Aifantis and NYU Department of Pathology will provide the resources critical to my training and research, ensuring my success. This extensive professional growth program will guide me during the mentored phase excelling as an independent academic scientist. Collectively, the proposed research and career development plans are expected to generate data with significant impact on circumventing targeted-therapy resistance in AML and setting the basis of my future research as an independent researcher.